Result from "Primer" programme

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Sun Nov 22 17:27:36 EST 1992


>I have been using the "Primer" programme on the published sequence of a mouse
>gene (HSP70.1) and it reports homology in the 3' non-transcribed region, with
>the mouse repetitve element B2.
>My question is, could this explain why my attempts at using a probe which
>is derived from the 3' region of this gene, has failed so far in Southern
>blots - all I get is hybridization to apparently all DNA, even when washed
>at 65 degrees in 0.1xSSC.
>Does anyone know HOW homolgous the sequence has to be before Primer reports
>it ?
>
>Any comments appreciated,
>Sean

Hi Sean

My guess is that the homology with the B2 element is your problem.  0.1xSSC
at 65 is certainly stringent enough to ensure that only true hybrids
remain.  However, in my experience even a 20bp region of total homology
with a repeat element is enough to turn your entire lane black.  Is it
possible to remove the region of homology?

I hope this helps.

Cheers Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

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